The Role And Mechanism Of Methyl-CpG-binding Domain 2 (MBD2) In The Pathogenesis Of Aortic Dissection

Background and objective:Aortic dissection(AD)is a life-threatening cardiovascular disease,which is formed by blood penetration into the medial layer,formation of a hematoma,and extended dissection along the aortic wall.The incidence of aortic dissection is increasing every year.In recent years,with the improvement of surgical intervention and medical equipment,its diagnosis and treatment have been greatly improved.However,the financial burden on aortic dissection patients and society is heavy.At present,the pathogenesis of aortic dissection is still not clear.Studies suggest that apoptosis is an essential pathological process for aortic dissection.Moreover,in recent years,it has been found that epigenetic DNA methylation mediates the process of AD vascular remodeling.Studies related to DNA methylation in cardiovascular disease are increasing year by year.Methyl-CpG-binding domain(MBD)protein is a family of molecules that interpret DNA methylation patterns.MBD2 is the molecule with the highest binding ability to methylated DNA in the MBD family and plays a crucial role in DNA methylation.Inhibition of MBD2 could protect the endothelial,tubular and retinal cells from apoptosis.Moreover,epigenetic DNA methylation mediates the inflammatory vascular remodeling process in AD.However,the relationship between MBD2 and AD has not reported,and the mechanism is still unclear.Therefore,this study aimed to explore the role and mechanism of MBD2 in the occurrence and development of aortic dissection,and provide a new intervention target and theoretical basis for the prevention and treatment of aortic dissection.Methods:(1)The aortic tissue of patients with aortic dissection and organ donors were collected(without aortic disease),preserved in liquid nitrogen and made paraffin blocks.The pathological changes of aortic tissue were observed by hematoxylin-eosin(HE)and elastic van Gieson(EVG)staining.The expression of MBD2 in the aortic tissue of organ donors and aortic dissection patients was detected by immunohistochemistry,Western Blot and real-time fluorescent quantitative PCR(RT-q PCR),and the expression location of MBD2 in aortic wall was detected by immunofluorescence staining.Then we identify the MBD2 gene knockout mice.The aortic dissection model of MBD2 gene knockout mice fed withβ-aminopropiononitrile(BAPN)and angiotensin II(Ang Ⅱ)micropump.Ultrasound was performed to detect the changes in the aortic diameter of mice,and observe the changes in the body weight of mice and the occurrence and rupture of aortic dissection.(2)TUNEL staining was used to detect the apoptosis of aortic tissue in patients with aortic dissection and mouse aortic dissection model.Western Blot was used to detect the changes of apoptotic proteins.HE and EVG staining were used to observe the pathological changes of aortic tissue in the MBD2 gene knockout mice’s aortic dissection model.Immunohistochemical method and Western Blot method were used to detect apoptosis of mouse aortic tissue.Additionally,Ang Ⅱ was used to stimulate human aortic vascular smooth muscle cells to establish a cell model of aortic dissection,MBD2 interference and overexpression cell lines were constructed.Then TUNEL staining,Western Blot and flow cytometry were used to detect the changes of apoptosis.(3)The tissue samples from patients with aortic dissection were sequenced and analyzed for biological information.The target genes that MBD2 can bind to and participate in the apoptosis process were screened.Western Blot and RT-q PCR were used to verify the expression of the target genes in human aortic dissection and mouse aortic dissection models.The expression of the target gene was observed in the aortic dissection model of MBD2 gene knockout mice.Ang Ⅱ stimulated aortic vascular smooth muscle cell model was constructed,and Western Blot was used to detect the expression of target genes.Chromatin immunoprecipitation(Ch IP)was used to verify the binding of MBD2 and the target gene,and methylated CpG-DNA Immunoprecipitation was used to detect the methylation of the target gene promoter.MBD2 interference and overexpression cell lines were constructed,and the target expression changes were detected by Western Blot.The target interference and overexpression cell lines were constructed,and the changes of apoptotic protein and apoptosis rate were observed by Western Blot and flow cytometry.Results:(1)Compared with the donor group,HE staining showed d HE staining showed loose and thickened and irregular aortic media in aortic dissection patients.EVG staining showed irregular shape of elastic fibers in aortic dissection tissue,and rupture.MBD2 was expressed increasingly in aortic dissection patients and localized in smooth muscle layer of aortic wall.In the mouse experiment,the modeling rate of aortic dissection in mice reached 90%.The expression of MBD2 increased in the mouse aortic dissection model,and the incidence of aortic dissection decreased after MBD2 gene knocked out.(2)Compared with donor group,apoptosis in aortic dissection group increased.In the mouse experiment,apoptosis increased in a mouse model of aortic dissection compared with the control group.Apoptosis in the aortic dissection model of MBD2 gene knockout mice was less than that in the mouse aortic dissection model group;Further VSMC experiments showed that the expression of MBD2 in Ang Ⅱ stimulated vascular smooth muscle cells(VSMC)was time and concentration dependent.Ang Ⅱ can promote the apoptosis of VSMC.Inhibiting the expression of MBD2 can inhibit the apoptosis of aortic dissection cell model.Overexpression of MBD2 can promote the apoptosis of aortic dissection cell model.(3)The whole transcriptome sequencing analysis showed that AIFM3 was highly expressed in patients with aortic dissection and involved in the process of apoptosis.The expression of AIFM3 is increased in patients with aortic dissection,mouse aortic dissection model and Ang Ⅱ stimulated aortic dissection cell model.Chromatin Immunoprecipitation(Ch IP)showed that MBD2 could bind to the AIFM3 promoter sequence in VSMC.Methylation analysis demonstrated that methylation of AIFM3 promoter region was reduced in aortic dissection cell model.Inhibition of MBD2 expression in aortic dissection cell model can reduce AIFM3 protein expression;Overexpression of MBD2 can increase the expression of AIFM3 in aortic dissection cell model.In addition,inhibition of AIFM3 expression in VSMC stimulated by Ang Ⅱ revealed that apoptosis was decreased.Overexpression of AIFM3 in aortic dissection cell model,the apoptosis was increased.Conclusions:MBD2 can bind the AIFM3 promoter CpG region,reduce methylation,increase apoptosis in aortic dissection cell model,which causes structural damage in the aortic membrane and lead to aortic dissection.